Project summary More than eight million people worldwide are infected by three closely related Kinetoplastid parasites: Trypanosoma brucei, Trypanosoma cruzi, and Leishmania. Among these, T. brucei causes fatal human African trypanosomiasis that threatens millions of people. Few treatments are available, and all have severe side effects. T. brucei evades its host's immune responses by regularly switching its major surface antigen, VSG. VSG is expressed from subtelomeric VSG expression sites (ESs) in a strict monoallelic manner, which also helps coordinate parasite proliferation and development. In addition, many microbial pathogens that undergo antigenic variation express their major surface antigens monoallelically. For example, in Plasmodium falciparum that causes deadly malaria in humans, monoallelic expression of var genes (many are at subtelomeric region) is essential for its antigenic variation. However, how monoallelic gene expression is achieved is not completely clear. We have shown that T. brucei RAP1, an essential telomere protein, silences VSGs in a telomere proximity-dependent manner, yet, many questions remain unanswered: How is RAP1 recruited to the telomere? How is RAP1 prevented from silencing the one expressed VSG? In Aim 1, we will determine whether the recently identified DNA binding activity of RAP1 and RAP1's interaction with known telomere binding factors help target RAP1 to the telomere. In Aim 2, we will determine whether the recently identified interaction between RAP1 and the active VSG RNA helps prevent RAP1 from silencing the active VSG. Elevated TERRA (the transcript of telomere repeats) expression in RAP1-depleted cells causes more DNA breaks at subtelomeric VSG loci, which is detrimental to cell viability. We will therefore examine how RAP1 regulates TERRA expression in Aim 3. We also found TERRA to be important for complete VSG silencing and will investigate its mechanisms in Aim 3. Finally, many factors participate in VSG ES expression regulation. How RAP1 coordinates with other ES regulators to achieve monoallelic VSG expression is unknown. We found that RAP1 interacts with a large scaffold protein, NOT1, whose yeast and human homologs are involved in gene expression regulation at multiple levels. We hypothesize that NOT1 mediates coordination between RAP1 and other ES regulators, which will be examined in Aim 4. Our proposed studies will reveal detailed mechanisms of RAP1-mediated VSG silencing and allelic exclusion VSG expression, with an emphasis on communications between active and silent VSG ESs, which is currently poorly studied. Based on our preliminary results, we expect to understand functions of RAP1 that are unique in T. brucei but not in mammalian hosts, which will help develop a means for eliminating T. brucei and other parasites employing similar antigenic variation mechanisms. As RAP1 is an essential telomere protein, knowledge gleaned from our studies will be easily extrapolated to closely related Kinetoplastid parasites and aid in their eradication.